1. Field of the Invention
The present invention relates to a γ-glutamylcysteine-producing yeast strain, to a method for culturing it, and to a food utilizing cells of that yeast strain. A material containing γ-glutamylcysteine and a material containing cysteine produced from γ-glutamylcysteine are useful in the field of foods.
2. Description of the Related Art
Cysteine is used for the purpose of enhancing the flavor of foods or the like. Known production methods of cysteine include, for example, proteolysis method and semi-synthetic method, and these methods are currently used in the main. Although natural food materials having high cysteine contents have been demanded for the purpose of using them to enhance the flavor of foods, such natural food materials have been rarely known. On the other hand, it has been reported that heat- or enzyme-treatment of yeast extracts containing γ-glutamylcysteine may give rise to food materials having high cysteine contents (WO 00/30474).
γ-glutamylcysteine is synthesized from cysteine and glutamic acid as substrates by the function of γ-glutamylcysteine synthetase. On the other hand, glutathione is synthesized from γ-glutamylcysteine and glycine as substrates by the function of glutathione synthetase. It has been reported that a yeast in which glutathione synthetase gene has been disrupted accumulates γ-glutamylcysteine (Otake et al., Agric. Biol. Chem., 54(12), 3145-3150, 1990).
Yeasts having high γ-glutamylcysteine contents have been reported in WO 00/30474, Otake et al., Agric. Biol. Chem., 54(12), 3145-3150, 1990, Chris et al., Molecular Biology of the Cell., 8, 1699-1707, 1997, Inoue et al., Biochimica et Biophysica Acta, 1395, 315-320, 1998, or the like. However, these reports has not disclosed the conditions for culturing the yeast, in which glutathione synthetase gene has been disrupted or weakened, to accumulate a large amount of γ-glutamylcysteine.
A method for culturing a yeast to accumulate a large amount of glutathione, a metabolic product of γ-glutamylcysteine, in its cells has been disclosed (JP 48-92579A and the like). This report describes that an amount of the accumulated glutathione has been increased when cysteine, a glutathione-constituting amino acid, was added during culturing a yeast. Therefore, it is considered that a large amount of γ-glutamylcysteine could be accumulated when cysteine is added during culturing the yeast in which glutathione synthetase gene is disrupted or weakened. However, it is not practical from an economical aspect to add cysteine during culturing a γ-glutamylcysteine-containing yeast for obtaining cysteine-containing materials, since γ-glutamylcysteine-containing materials are useful for producing cysteine-containing materials.
Further, Otake et al. has reported the γ-glutamylcysteine contents in the cells of the yeast YL1 strain, in which a glutathione synthetase gene has been disrupted, when 3 mM of cysteine was added during culturing the yeast (Otake et al., Agri. Biol. Chem., 54(12), 3145-3150, 1990). This report describes that the amount of the accumulated γ-glutamylcysteine was 0.533% when the YL1 strain was cultured in the presence of cysteine, while the amount was 0.518% when the strain was cultured in the absence of cysteine. This result implies that it is not practical to add cysteine during culturing the yeast in which glutathione synthetase gene has been disrupted or weakened.
It has also been reported that glutathione contents in yeast cells has increased when expression of a MET25 gene was enhanced. Further, as a method for increasing expression of the MET25 gene, a method by utilizing a mutant MET4 gene (Omura et al., FEBS Letters 387(1996) 179-183 and JP 10-33161A) and a method by utilizing a mutant MET30 gene (DOMINIQUE et al., MOLECULAR AND CELLUAR BIOLOGY, December 1995, p6526-6534) have been reported.
The mechanism of the expression of the MET25 gene is considered as follows. That is, the MET4 gene product functions as a positive regulator for the expression of the MET25 gene. In general, the MET4 gene product forms a SCFMET30 complex together with the MET30 gene product and other several proteins, and the MET4 gene product is ubiquitinated and decomposed together with the MET30 gene product by a proteolytic system of 26S proteasome, thereby, the expression of the MET25 gene is suppressed. On the other hand, when the function of the SCFMET30 complex is deteriorated, the MET4 gene product and the MET30 gene product are not decomposed and the MET25 gene is expressed (Patton et al., Genes Dev. 12: 692-705, 1998 and Rouillon et al., EMBO Journal 19: 282-294, 2000).
Based on these reports, it is suggested that γ-glutamylcysteine content could also be increased in a yeast having a high γ-glutamylcysteine content by enhancing the expression of the MET25 gene.
In addition, it has been reported that when “sake” yeast was cultured in a calcium panthotenate-deficient condition, the yeast has accumulated hydrogen sulfide in its logarithmic growth phase. This report pays its attention to the generation of hydrogen sulfide from cysteine, and it also describes that the phenomenon is further promoted in a pantothenic acid-deficient condition.